Variability of genotype phenotypic expression of spinal muscular atrophy in patients from Serbia

[M. D. Mišković]

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Biologija - Medicinska genetika / Biology - Medical genetics

Spinalna mišićna atrofija (SMA) je drugo najčešće autozomno recesivno oboljenje kod ljudi. Prema uzrastu kada se pojavljuju prvi simptomi i težini kliničke slike SMA je klasifikovana u tri tipa. Mutacije u telomernoj kopiji gena SMN1 (eng. survival of motor neuron), koji se nalazi u 5q13 regionu, dovode do nastanka bolesti, dok ostali geni u ovom regionu predstavljaju potencijalne modifikatore SMA fenotipa. Detekcija homozigotne delecije egzona 7 i 8 gena SMN1, primenom PCR amplifikacije i polimorfizma dužine restrikcionih fragmenata (PCR/RFLP, eng. Polymerase Chain Reaction/Restriction Fragment Length Polymorphism) urađena je na uzorcima 107 SMA pacijenata iz Srbije i 100 kontrolnih uzoraka DNK poreklom od zdravih osoba. Kod pacijenata bez homozigotne delecije kod kojih je klinička reevaluacija potvrdila dijagnozu SMA je primenom multipleks ligaciono-zavisne amplifikacije proba (MLPA, eng. Multiplex Ligation-dependent Probe Amplification) određivan broj kopija prisutnog gena SMN1 u cilju detekcije složenih heterozigotnih nosilaca delecije na jednom i tačkaste mutacije na drugom hromozomu. Kod potvrđenih heterozigota sekvenciran je egzon 6 gena SMN u cilju detekcije intragenskih mutacija. Za ispitivanje varijabilnosti genotipa 5q13 regiona i njegove povezanosti sa fenotipskom ekspresijom bolesti prvo je na istom uzorku analizirana homozigotna delecija gena NAIP (eng. neuronal apoptosis inhibitory protein). Nakon toga, primenom MLPA metode je kod 30 pacijenata sa prethodno dijagnostikovanom homozigotnom delecijom gena SMN1 određivan broj kopija gena iz 5q13 regiona: NAIP, SMN2 (centromerna kopija gena SMN), GTF2H2 (eng. general transcription factor IIH subunit 2) i gen koji kodira protein nepoznate funkcije - H4F5. Studija je obuhvatila i ispitivanje 39 zdravih osoba sa porodičnom SMA anamnezom kod kojih je analizom broja kopija gena SMN1 određivan status heterozigotnih nosilaca delecije ovog gena. Kod 66 fetusa iz 44 porodice sa visokim rizikom za dobijanje obolelog potomstva sprovedena je prenatalna dijagnostika za direktnu detekciju homozigotne SMN1 delecije. Homozigotna delecija gena SMN1 je uočena kod 81% (87/107) ipitivanih SMA pacijenata: delecija egzona 7 i 8 kod 76,6% (82/107) i delecija samo egzona 7 kod 4,7% (5/107). Učestalost homozigotne delecije bila je najveća u grupi pacijenata sa SMA tip I - 93,1% (54/58), u odnosu na SMA tip II - 71,4% (25/35) i SMA tip III - 57,1% (8/14). Kod 23,1% (3/13) pacijenta bez homozigotne delecije gena SMN1 kod kojih je klinička reevaluacija potvrdila dijagnozu SMA detektovana je jedna SMN1 kopija i potvrđena je heterozigotnost...

Spinal muscular atrophy (SMA) is the second most frequent autosomal recessive disease in humans. According to the age of onset and severity of clinical manifestations, SMA is classified into three types. Mutations in the telomeric copy of the survival of motor neuron gene (SMN1) cause the disease while other genes in 5q13 region has been considered as modifying factors of the SMA severity. Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/RFLP) method was used for direct detection of homozygous deletions of SMN1 exons 7 and 8 in 107 SMA patients and 100 healthy controls from Serbia. Patients without homozygous SMN1 deletion in which clinical re-evaluation confirmed SMA diagnosis were further analysed using Multiplex Ligation-dependent Probe Amplification (MLPA) for determination of SMN1 copy number to identify compound heterozygotes of deletion and point mutation. Sequencing of SMN exon 6 for detection of subtle mutations was performed in patients with confirmed heterozygosity. For examination of genotype variability in 5q13 region and its correlation with phenotype homozygous deletion of neuronal apoptosis inhibitory protein (NAIP) gene was first analysed using PCR in the same sample of patients and controls. After that, 30 patients with homozygous SMN1 deletion were further examined using MLPA to determine copy number of genes in 5q13 region: NAIP, centromeric copy of SMN gene (SMN2), general transcription factor IIH subunit 2 (GTF2H2) and putative RNA-binding protein (H4F5). The study also included the analysis of SMN1 copy number for carrier status determination in 39 healthy individuals with SMA history. Prenatal diagnosis for direct detection of homozygous SMN1 gene deletion was performed on 66 fetal samples from 44 families at high SMA risk. Homozygous SMN1 gene deletion was detected in 81.3% (87/107) of SMA patients: deletion of exons 7 and 8 in 76.6% (82/107), deletion of exon 7 in 4.7% (5/107). The highest deletion frequency was among SMA type I patients – 93.1% (54/58) compared to SMA type II – 71.4% (25/35) and SMA type III – 57.1% (8/14). One SMN1 gene copy was detected in 23.1% (3/13) patients without homozygous deletion of this gene and with clinically reassessed confirmation of SMA diagnosis and heterozygosity was confirmed. Among them two patients, classified as SMA type II, had missense mutation c.821C>T. Homozygous deletion of NAIP gene was present in 21.5% (23/107) of SMA patients and 1% (1/100) of controls...

SMA, SMN1, NAIP, SMN2, GTF2H2, H4F5, homozigotna delecija, broj kopija, status heterozigotnog nosica

SMA, SMN1, NAIP, SMN2, GTF2H2, H4F5, homozygous deletion, copy number, carrier status

616.8-003.99:[575.224.2:575.21/.22](043.3)

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